Isolated peptides which bind to MHC Class II molecules, and uses thereof

ABSTRACT

Peptides which have an amino acid sequence identical to sequences found in tumor rejection antigen precursors, such as NY-ESO-1, and SSX-2, are disclosed. These peptides bind to MHC-Class II molecules, such as HLA-DR molecules, and provoke proliferation of CD4+ cells.

RELATED APPLICATIONS

This application is a divisional application of Ser. No. 09/408,036filed Sep. 29, 1999, now U.S. Pat. No. 6,800,730, which is acontinuation in part of Ser. No. 09/165,546, filed on Oct. 2, 1998, nowU.S. Pat. No. 6,723,832 and Ser. No. 09/344,040, filed on Jun. 25, 1999,now U.S. Pat. No. 6,548,064, all of which are incorporated herein byreference.

FIELD OF THE INVENTION

This invention relates to HLA binding peptides derived from antigensassociated with cancer. These peptides bind to Class II MHC molecules.

BACKGROUND AND PRIOR ART

It is fairly well established that many pathological conditions, such asinfections, cancer, autoimmune disorders, etc., are characterized by theinappropriate expression of certain molecules. These molecules thusserve as “markers” for a particular pathological or abnormal condition.Apart from their use as diagnostic “targets”, i.e., materials to beidentified to diagnose these abnormal conditions, the molecules serve asreagents which can be used to generate diagnostic and/or therapeuticagents. A by no means limiting example of this is the use of cancermarkers to produce antibodies specific to a particular marker. Yetanother non-limiting example is the use of a peptide which complexeswith an MHC molecule, to generate cytolytic T cells against abnormalcells.

Preparation of such materials, of course, presupposes a source of thereagents used to generate these. Purification from cells is onelaborious, far from sure method of doing so. Another preferred method isthe isolation of nucleic acid molecules which encode a particularmarker, followed by the use of the isolated encoding molecule to expressthe desired molecule.

To date, two strategies have been employed for the detection of suchantigens, in e.g., human tumors. These will be referred to as thegenetic approach and the biochemical approach. The genetic approach isexemplified by, e.g., dePlaen et al., Proc. Natl. Sci. USA 85: 2275(1988), incorporated by reference. In this approach, several hundredpools of plasmids of a cDNA library obtained from a tumor aretransfected into recipient cells, such as COS cells, or intoantigen-negative variants of tumor cell lines which are tested for theexpression of the specific antigen. The biochemical approach,exemplified by, e.g., O. Mandelboim, et al., Nature 369: 69 (1994)incorporated by reference, is based on acidic elution of peptides whichhave bound to MHC-class I molecules of tumor cells, followed byreversed-phase high performance liquid chromography (HPLC). Antigenicpeptides are identified after they bind to empty MHC-class I moleculesof mutant cell lines, defective in antigen processing, and inducespecific reactions with cytotoxic T-lymphocytes. These reactions includeinduction of CTL proliferation, TNF release, and lysis of target cells,measurable in an MTT assay, or a ⁵¹Cr release assay.

These two approaches to the molecular definition of antigens have thefollowing disadvantages: first, they are enormously cumbersome,time-consuming and expensive; and second, they depend on theestablishment of cytotoxic T cell lines (CTLs) with predefinedspecificity.

The problems inherent to the two known approaches for the identificationand molecular definition of antigens is best demonstrated by the factthat both methods have, so far, succeeded in defining only very few newantigens in human tumors. See, e.g., van der Bruggen et al., Science254: 1643-1647 (1991); Brichard et al., J. Exp. Med. 178: 489-495(1993); Coulie, et al., J. Exp. Med. 180: 35-42 (1994); Kawakami, etal., Proc. Natl. Acad. Sci. USA 91: 3515-3519 (1994).

Further, the methodologies described rely on the availability ofestablished, permanent cell lines of the cancer type underconsideration. It is very difficult to establish cell lines from certaincancer types, as is shown by, e.g., Oettgen, et al., Immunol. Allerg.Clin. North. Am. 10: 607-637 (1990). It is also known that someepithelial cell type cancers are poorly susceptible to CTLs in vitro,precluding routine analysis. These problems have stimulated the art todevelop additional methodologies for identifying cancer associatedantigens.

One key methodology is described by Sahin, et al., Proc. Natl. Acad.Sci. USA 92: 11810-11913 (1995), incorporated by reference. Also, seeU.S. Pat. No. 5,698,396, and patent application Ser. No. 08/479,328filed Jan. 3, 1996. All three of these references are incorporated byreference. To summarize, the method involves the expression of cDNAlibraries in a prokaryotic host. (The libraries are secured from a tumorsample). The expressed libraries are then immunoscreened with absorbedand diluted sera, in order to detect those antigens which elicit hightiter humoral responses. This methodology is known as the SEREX method(“Serological identification of antigens by Recombinant ExpressionCloning”). The methodology has been employed to confirm expression ofpreviously identified tumor associated antigens, as well as to detectnew ones. See the above referenced patent applications and Sahin, etal., supra, as well as Crew, et al., EMBO J 144: 2333-2340 (1995).

The SEREX methodology has been employed in a number of instances toidentify cancer associated antigens. See, e.g., PCT/US99/06875,describing a cancer associated antigen found to be expressed by, interalia, esophageal cancer and melanoma. This antigen is referred to asNY-ESO-1. See U.S. Pat. No. 5,804,381 as well as Chen, et al, Proc.Natl. Acad Sci USA—92:8125-8129 (1995). Additionally, a family ofrelated antigens, the “SSX” family, has been identified using thismethodology. See PCT/US99/14493 and Ser. No. 09/105,839 filed Jun. 26,1998 in this regard.

Following the identification of full length molecules as cancerassociated antigens, the next step has been to identify those portionsof the antigens which are relevant as binding partners for MHC or HLAmolecules. The resulting complexes serve as targets for identificationby T cells, which then proliferate and eliminated the cells whichpresent such complexes.

Early work focused on the identification of those peptide moleculeswhich bind to Class I molecules stimulating proliferation of CD8⁺ Tcells. See, e.g., U.S. Pat. No. 5,925,729, which shows this for onefamily of antigens. Also see PCT/US99/06875 and PCT/US99/14493 forfurther work on the identification of peptides which bind to MHCmolecules. All of these are incorporated by reference.

The presence of antibodies against a particular molecule suggests that aprocess other than presentation by MHC Class I molecules is involved. InPCT/US99/06875, supra, evidence is presented showing that the NY-ESO-1molecule is processed to peptides which are presented by MHC Class IImolecules.

This work has been continued. The disclosure which follows shows thatadditional peptides have been identified which bind to MHC Class IImolecules, and stimulate proliferation of CD4⁺ T cells. These peptidesare derived from both NY-ESO-1 and SSX-2. These, and other features ofthe invention, are set forth in the disclosure which follows.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the expression pattern of RNA for the NY-ESO-1 antigen, invarious tissue types.

FIG. 2 shows Northern Blot analysis of NY-ESO-1 mRNA, which was found intestis and cell line SK-MEL-19, but not in various other cell and tissuesamples.

FIG. 3 shows potential sites for modification of the deduced amino acidsequence of NY-ESO-1.

FIG. 4 is a hydrophilicity plot of NY-ESO-1, showing hydrophilic domainsin the amino terminus and a long, hydrophobic stretch close to thecarboxyl end.

FIG. 5 shows the results of CTL lysis studies using various cells whichare HLA-A2 positive, NY-ESO-1 positive, positive for both, or positivefor neither.

FIG. 6 presents data establishing that HLA-A2 is the presenting moleculefor presentation of SEQ ID NO: 1 derived peptides.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS EXAMPLE 1

Total RNA was extracted from a snap frozen specimen of well tomoderately differentiated squamous cell cancer of the esophagus, usingwell known methods. See, e.g., Chomzynski, J. Analyt. Biochem. 162:156-159 (1987), for one such method. This RNA was used to prepare a cDNAlibrary which was then transfected into λZAP phage vectors, inaccordance with the manufacturer's instructions. The λZAP library wasthen transfected into E. coli, yielding 1.6×10⁶ primary isolates.

The SEREX methodology of Sahin, et al., Proc. Natl. Acad. Sci. USA 92:11810-11813 (1995), incorporated by reference, was then used. In brief,autologous serum was stripped of antibodies against molecules which areendogenous to E. coli by combining the serum with lysates of E. colitransfected with phage λZAP which did not contain the cDNA clones fromthe esophageal cancer cells.

The depleted serum was then diluted, and mixed with nitrocellulosemembranes containing phage plaques. The plaques were incubatedovernight, at room temperature. Washing followed, and then the filterswere incubated with alkaline phosphatase conjugated goat anti human FCγsecondary antibodies, and reactive phage plaques were visualized byincubating with 5-bromo-4-chloro-indolyl phosphate and nitrobluetetrazolium. A total of 13 positive clones were found.

EXAMPLE 2

Following identification, the reactive clones were subcloned tomonoclonality via dilution cloning and testing with human serum. Theseclones were then purified, excised in vitro, and converted into pBK-CMVplasmid forms, using the manufacturer's instructions. The inserted DNAwas then evaluated using EcoRI-XbaI restriction mapping to determinedifferent inserts. Eight different inserts were identified, ranging insize from about 500 to about 1.3 kilobase pairs. The clones weresequenced using an ABI PRISM automated sequencer.

Table 1 summarizes the results. One gene was represented by fouroverlapping clones, a second by three overlapping clones, and theremaining six by one clone only.

A homology search revealed that the clones referred to as NY-ESO-2, 3,6, 7 were already known. See Elisei, et al., J. Endocrin. Invest. 16:533-540 (1993); Spritz, et al., Nucl. Acids Res. 15: 10373-10391 (1987);Rabbits, et al., Nature Genetics 4: 175-180 (1993); Crozat, et al.,Nature 363: 640-644 (1993); GenBank H18368 and D25606. Two of the clones(NY-ESO-3 and NY-ESO-6), have previously been shown to be expressed invarious normal human tissues. No evidence of lineage restriction hasbeen found. NY-ESO-6 (cDNA), appears to be the 3′-untranslated portionof the FUS/TLS gene. In experiments not reported here, sequencing andSouthern Blot analysis of NY-ESO-6 showed no evidence of translocationor point mutations in the cancer. Four of the clones, i.e., NY-ESO-1, 4,5 and 8 showed no strong homology to sequences in the databasesexamined, and were thus studied further.

TABLE 1 Genes isolated from esophageal cancer library by immunoscreeningwith autologous serum GENE CLONE# Size DNA databank Comments NY-ESO-1E1-5b 679 bp No strong homology expressed in testis and E1-114b 614 bpovary E1-153c 670 bp E1-50 679 bp NY-ESO-2 E1-71a 605 bp U1 smallnuclear cloned by Ab screening E1-140 874 bp RNP 1 homolog (thyroiditispatient) E1-31 750 bp NY-ESO-3 E1-141b 517 bp Colon 3′ direct Mbol (dbjD25606, gb H18638) cDNA; Adult brain unpublished cDNA NY-ESO-4 E1A-10c400 bp No strong homology ubiquitous expression in normal tissuesNY-ESO-5 E1A-54 670 bp No strong homology expressed in normal esophagusNY-ESO-6 E1B-9b −1.2 kb Human fus mRNA translocated in liposarcoma t(12;16) NY-ESO-7 E1B-20f −1.0 kb human U1-70k sn RNP different from NY-ESO-2(embl HSU17052, gbM22636) NY-ESO-8 E1B-20g −1.3 kb No strong homologyubiquitous expression in normal tissues

EXAMPLE 3

Studies were carried out to evaluate mRNA expression of the NY-ESO 1, 4,5 and 8 clones. To do this, specific oligonucleotide primers weredesigned for each sequence, such that cDNA segments of 300-400 basepairs could be amplified, and so that the primer melting temperaturewould be in the range of 65-70° C. Reverse transcription-PCR was thencarried out using commercially available materials and standardprotocols. A variety of normal and tumor cell types were tested. Theclones NY-ESO-4 and NY-ESO-8 were ubiquitous, and were not studiedfurther. NY-ESO-5 showed high level expression in the original tumor,and in normal esophageal tissue, suggesting that it was adifferentiation marker.

NY-ESO-1 was found to be expressed in tumor mRNA and in testis, but notnormal colon, kidney, liver or brain tissue. This pattern of expressionis consistent with other tumor rejection antigen precursors.

EXAMPLE 4

The RT-PCR assay set forth supra was carried out for NY-ESO-1 over amuch more complete set of normal and tumor tissues. Tables 2, 3 and 4show these results. In brief, NY-ESO-1 was found to be highly expressedin normal testis and ovary cells. Small amounts of RT-PCR productionwere found in normal uterine myometrium, and not endometrium, but thepositive showing was not consistent. Squamous epithelium of various celltypes, including normal esophagus and skin, were also negative.

When tumors of unrelated cell lineage were tested, 2 of 11 melanomascell lines showed strong expression, as did 16 of 67 melanoma specimens,6 of 33 breast cancer specimens and 4 of 4 bladder cancer. There wassporadic expression in other tumor types.

TABLE 2 mRNA distribution of NY-ESO-1 in normal tissues Tissue mRNATissue mRNA Esophagus − Adrenal − Brain* − Pancreas − Fetal Brain −Seminal Vesicle − Heart − Placenta − Lung − Thymus − Liver − Lymph node− Spleen − Tonsil − Kidney − PBL − Stomach − PBL, activated# − Smallintestine − Melanocytes − Colon − Thyroid − Rectum − Uterus +/−** Breast− Testis + Skin − Ovary + *tissues from several parts tested with IL-2and PHA **weakly positive in some specimens, negative by Northern blot

TABLE 3 mRNA distribution of NY-ESO-1 in melanoma and breast cancer celllines: Cell line NY-ESO-1 mRNA MZ2-MEL3.1 − MZ2-MEL2.2 − SK-MEL-13 −SK-MEL-19 + SK-MEL-23 − SK-MEL-29 − SK-MEL-30 − SK-MEL-31 − SK-MEL-33 −SK-MEL-37 + SK-MEL-179 − SK-BR-3 − SK-BR-5 − 734B − MDA-MB-231 −

TABLE 4 NY-ESO-1 mRNA expression in various human tumors by RT-PCR mRNAmRNA tumor type (positive/total) tumor type (positive/total) melanoma25/77 ovarian cancer 2/8 breast cancer 17/43 thyroid cancer 2/5 prostatecancer  4/16 bladder cancer  9/13 colon cancer  0/16 Burkitt's lymphoma1/2 glioma  0/15 basal cell carcinoma 0/2 gastric cancer  0/12Jejomyosarcoma 0/2 lung cancer  5/17 other sarcomas 0/2 renal cancer 0/10 pancreatic cancer 0/2 lymphoma*  0/10 seminoma 0/1 hepatoma 2/7spinal cord tumor 0/1 *non-Hodgkin's, non-Burkitt's types.

A further set of experiments were carried out to ascertain if thepresence of anti NY-ESO-1 antibody in cancer patient sera could bedetermined via an ELISA.

To elaborate, recombinant NY-ESO-1 in a solution of coating buffer (15mM Na₂CO₃, 30 mM NaHCO₃, pH 9.6, 0.02% NaN₃), at a concentration of 1ug/ml, was adsorbed to microwell plates (10 ul of solution per well),and then kept overnight at 4° C. The plates were washed with phosphatebuffered saline, and blocked, overnight, at 4° C., with 10 ul/well of 2%bovine serum albumin/phosphate buffered saline. After washing, 10ul/well of diluted serum in 2% bovine serum albumin was added to thewells. Following two hours of incubation at room temperature, plateswere washed, and 10 ul/well of goat anti-human IgG-alkaline phosphataseconjugates were added, at a 1:1500 dilution. This solution was incubatedfor one hour at room temperature, followed by washing and addition of asolution of substrate for the alkaline phosphatase (10 ul/well). After25 minutes at room temperature, the wells were read with a fluorescenceplate reader. The results are presented in the following table:

Cancer patients: Eso 1+/total tested % melanoma  12/127 9.4 ovariancancer  4/32 12.5 lung cancer  1/24 4.0 breast cancer  2/26 7.7 Blooddonors  0/70 0

In order to determine whether there was a relationship betweenexpression of mRNA for NY-ESO-1 in tumors, and antibody response to theNY-ESO-1 protein, data from sixty-two melanoma patients were compared.All patients whose serum was reactive with NY-ESO-1 protein (i.e.,contained antibodies to NY-ESO-1), also had NY-ESO-1 positive tumors,while no patients with NY-ESO-1 negative tumors showed antibodies toNY-ESO-1 in their serum. There was a percentage of NY-ESO-1 positivepatients who lacked the antibody. Given that about 20-40% of melanomasexpressed NY-ESO-1, and only patients with NY-ESO-1 positive tumors haveantibody, the data suggest a high percentage of patients with NY-ESO-1positive tumors develops antibodies against the protein, thus suggestinga broad scale assay useful in diagnosis and responsiveness to treatment.

EXAMPLE 5

Northern blot analysis was then carried out to investigate the size ofthe NY-ESO-1 transcript, and to confirm tissue expression patterns. Themethodology of Ausubel, et al., Current Protocols In Molecular Biology(John Wiley & Sons, 1995) was used. To be specific, 20 ug of total RNAper lane were dissolved in a formamide and formaldehyde containingbuffer, heated to 65° C., and then separated on a 1.2% agarose gel, with3% formaldehyde, followed by transfer to nitrocellulose paper.Hybridization was then carried out using a ³²P labelled probe, followedby high stringency washing. The final wash was at 0.1×SSC, 0.1% SDS, 60°C., for 15 minutes.

RNA from testis, and a melanoma cell line (SK-MEL-19) which had beenpositive for NY-ESO-1 in the prior assays, showed an RNA transcript ofabout 0.8-0.9 kb. An esophageal carcinoma specimen showed a smear in the0.4-0.9 kb range, reflecting partial degradation. RNA from additionaltissues or cell lines tested showed no transcript.

To get cDNA encoding the full transcript, the esophageal cDNA librarywas rescreened, using plaque hybridization, and the original cDNA cloneas the hybridization probe. When 3×10⁵ clones were screened, sixpositives were found. The three longest clones were sequenced. Analysisof open reading frames showed that all three contained the entire codingregion, and 5′-untranslated regions of variable size. The longest clone,755 base pairs in length, (excluding polyA), contains a 543 base paircoding region, together with 53 untranslated bases at the 5′ end and 151untranslated base pairs at the 3′-end. See SEQ ID NO: 1 (also, FIG. 3).

The long ORF indicated that the deduced sequence of NY-ESO-1 protein is180 amino acids. The single immunopositive clone contained a sequenceencoding 173 of these. Deduced molecular mass is 17,995 daltons.

Analysis shows that there is an abundance of glycine residues in theN-terminal portion (30 of the first 80, 4 in the remaining 100).Hydrophilicity analysis indicated that there were hydrophilic antigenicsequences in the N-terminal half of the molecule, with alternatinghydrophobic and hydrophilic sequences, ending with a long, C-terminalhydrophobic tail (amino acids 152-172), followed by a short hydrophilictail. This pattern suggests a transmembrane domain. There are severalpotential N-myristorylation sites, 3 phosphorylation sites, and noevidence of N-glycosylation sites.

EXAMPLE 6

A melanoma cell line “NW-MEL-38” was established, in 1995, from apatient who suffered from malignant melanoma. Serum samples, peripheralblood lymphocytes, and tumor samples, were taken from the subject andfrozen, until the work described herein was carried out. In anticipationof evaluating antitumor T cell response in this patient, the patient wasHLA typed as HLA-A1 and HLA-A2.

To determine whether melanoma from this patient expressed NY-ESO-1,total RNA was isolated from both tumor samples and cell line NW-MEL-38,using standard techniques. Then, two micrograms of the total RNA, fromeach samples were subjected to cDNA synthesis, again using standardtechniques.

The cDNA was then used in RT-PCR experiments, using the followingprimers:

(SEQ ID NO: 1), 5′-CACACAGGAT CCATGGATGC TGCAGATGCG G′-3′, and (SEQ IDNO: 3) CACACAAAGC TTGGCTTAGC GCCTCTGCCC TG-3′These primers should amplify a segment of SEQ ID NO: 1 which spansnucleotides 271 to 599.

Amplification was carried out over 35 cycles, using an annealingtemperature of 60° C. The PCR products were visualized via ethidiumbromide staining, on a 1.5% agarose gel.

The results indicated that both the tumor and the cell line expressedSEQ ID NO: 1. The cell line and tumor samples were used in subsequentexperiments.

EXAMPLE 7

The isolated cDNA molecule, discussed supra, was then used to makerecombinant protein. Specifically, the cDNA was PCR amplified, usingstandard techniques, and was then cloned into a commercially availableplasmid vector, i.e., pQE9, which contains His tags. In work notelaborated upon herein, a second vector, pQE9K was also used. Thisdiffers from PQE9 in that kanamycin resistance is imparted by pQE9K,rather than ampicillin resistance.

The plasmid vector was transformed into E. coli strain XL1-Blue, andpositive transformants were identified via restriction mapping and DNAsequencing. Production of recombinant protein was induced usingisopropyl β-D-thiogalactoside, and the protein was purified on an Ni²⁺ion chromatography column, following well known procedures. The proteinwhen analyzed via 15% SDS-PAGE and silver staining, was identified as aprotein with a molecular weight of about 22 kilodaltons. This isconsistent with the anticipated size of the protein from its sequence.Two other forms of the recombinant protein were also identified. Theseconsisted of amino acids 10-180, and 10-121 of the amino acid sequencereported in SEQ ID NO: 1. They have molecular weights of about 14 kD and20 kD, respectively, on SDS-PAGE, as carried out supra.

An additional set of experiments were carried out to express NY-ESO-1 inbaculovirus. To elaborate, the NY-ESO-1 cDNA insert was released fromthe pQE9 vector, by cleavage with BamHI and HindIII. This insert wasthen subcloned into a commercially available baculovirus vector whichhad been cleaved with the same enzymes. Positive clones were determined,using standard methods, and transfected into recipient Sf9 cells.Recombinant viruses were then used to infect insect cells, using astandard medium (IPL-41), supplemented with 10% fetal calf serum. Themultiplicity of infection for the work was 20. Expression of recombinantprotein was determined as described supra. The recombinant proteinproduced in this vector carries an His-tag, so it was purified on Ni²⁺affinity columns, also as described, supra. The protein consists ofamino acids 10-180, and has a molecular weight of 20 kD via SDS-PAGE.

Additional eukaryotic transfectants were then produced. To do this, theNY-ESO-1 coding sequence was isolated from the pQE9 vector describedsupra, and then cloned into BamHI-HindIII sites of eukaryotic expressionvector pcDNA 3.1. Next, COS-7 cells were transfected with this vector,by contacting cell samples with 150 ng of the plasmid discussed supra,and 150 ng of plasmid pcDNA 1 Amp, which contained either cDNA forHLA-A2.1 or cDNA for HLA-A1, The well known DEAE-dextran chloroquinemethod was used. The cells were then incubated at 37° C., for 48 hours,after which they were tested in a CTL stimulation assay. Specifically,the assay followed Traversari et al, Immunogenetics 35: 145-148 (1992),incorporated by reference. In brief, 2500 CTLs, (NW38-IVS-1, see example9, infra), in 100 ul RPMI supplemented with 100% human serum, and 25U/ml of recombinant IL-2 were added to microwells containing COS-7transfectants (20,000 cells/well). After 24 hours, 50 ul of supernatantwere collected from each well, and TNF-α levels were determined in astandard assay, i.e., one where cytotoxicity against WEHI 164 clone 13cells were tested, using MTT. Positive cells were used in the WesternBlot analysis, described in the example which follows.

The CTLs used were CTL NW38-IVS-1, prepared in accordance with Knuth etal., Proc. Natl. Acad. Sci. USA 81: 3511-3515 (1984), incorporated byreference. Specifically, mixed lymphocyte T cell cultures were set up,by combining 10⁵ autologous NW38 MEL-1 tumor cells, and 10⁶ peripheralblood lymphocytes, taken from the subject. The cytokine IL-2 was added,and the mixed culture was incubated for one week at 37° C. Tumor cellswere removed, and a new aliquot of 5×10⁴ tumor cells were added togetherwith IL-2. This process was repeated weekly, until a strong response wasseen when tested against ⁵¹Cr labelled NW-MEL-38 cells. The responder Tcells were collected and frozen until used in further experiments.

EXAMPLE 8

Western Blot analysis was then carried out, using the serum samplesdescribed supra, as well as cell lysates taken from the cell lineNW-MEL-38, described supra, and the COS-7 transfectants, describedsupra, and the purified recombinant protein, also described supra. Serumsamples were taken from various points of the patient's therapy. Therewas no difference in the results.

In these assays, 1 ug of recombinant NY-ESO-1 protein, or 5 ul of celllysates of either type were diluted in SDS and boiled for five minutes,and then electrophoresed on a 15% SDS gel. After overnight blotting onnitrocellulose (0.45 um), and blocking with 3% BSA, the blots wereincubated with serum, diluted at 1:1000, 1:10,000, and 1:100,000, orwith a monoclonal antibody against NY-ESO-1, diluted to 1:50, as apositive control. The monoclonal antibody was prepared via Chen, et al.,Proc. Natl. Acad. Sci. USA 5915-5919 (1996), incorporated by referenceand elaborated as follows. BALB/C mice were immunized via fivesubcutaneous injections of recombinant NY-ESO-1 protein, at 2-3 weekintervals. The immunizing formulation included 50 ug of recombinantprotein in adjuvant. The first injection used Complete Freund'sAdjuvant, and Incomplete Freund's Adjuvant was used thereafter. Spleencells were taken from the immunized mice, and fused with mouse myelomacell line SP2/0, to generate hybridomas. Representative hybridoma E978was used for generation of mAbs.

Once hybridomas were generated, they were cloned, and their supernatantswere screened against recombinant protein, using a standard solid phaseELISA on microtiter plates. The assay was in accordance with Dippold etal., Proc. Natl. Acad. Sci. USA 77: 6114-6118 (1980), incorporated byreference. A series of negative controls were also run, usingrecombinant NY-ESO-1. Serum antibodies which bound to recombinantprotein, produced by E. coli as described, supra were visualized usinggoat anti-human IgG, labelled with alkaline phosphatase at 1:10,000dilution, and were then visualized with NBT-phosphate. UntransfectedCOS-7 cells were also used as a control. Serum from a healthy individualwas also used as a control.

Strong reactivity against the recombinant protein was found at serumdilutions down to 1:100,000, and there was also reactivity againstlysate of NW-MEL-38. There was no reactivity found against theuntransfected COS-7 cells, nor did the serum from a healthy individualshow reactivity.

EXAMPLE 9

Four different forms of NY-ESO-1 are described supra, i.e., the formproduced by SEQ ID NO: 1 in E. coli, as well as one consisting of aminoacids 10-180, one consisting of amino acids 10-121, and a form,expressed in the baculovirus vector system discussed supra whichconsisted of amino acids 10-180. Each form was used in ELISAs, followingthe above described protocols. All forms of the protein were found to beequally reactive with antibodies taken from various patients, as well asthe murine monoclonal antibodies discussed, supra.

EXAMPLE 10

In the testing of the COS-7 transfectants, supra, and the assaysdiscussed in this example, a cytolytic T cell line “NW38-IVS-1” wasused. This “CTL” was generated, via in vitro stimulation of theperipheral blood lymphocytes mentioned supra, using the tumor cell lineNW-MEL-38. This was done using standard techniques.

The CTL was used in a cytotoxicity assay with NW-MEL-38 (which wasHLA-A1, A2 positive, and NY-ESO-1 positive), along with two allogeneiccell lines which were NY-ESO-1 and HLA-A2 positive (SK-MEL-37 andMZ-MEL-19), a cell line which is MHC Class I negative (SK-MEL-19), acell line which is HLA-A2 positive, but NY-ESO-1 negative (NW-MEL-145),along with control cell lines K562 and autologous phytohemagglutininstimulated blasts. Various effector/target ratios were used, and lysisof ⁵¹Cr labelled target cells was the parameter measured. FIG. 5 showsthis.

The results indicated that the CTL NW38-IVS-1 lysed both the autologouscell line NW MEL-38, and the allogeneic cell lines which were HLA-A2 andESO-1 positive. Hence, the CTL was reactive with allogeneic materials.See FIG. 6.

EXAMPLE 11

As patient NW38 was HLA-A1 and HLA-A2 positive, experiments were carriedout to determine which MHC molecule was the presenting molecule.

The same experiment, described supra with COS-7 cells was carried out,except that, in these experiments, care was taken to secure separategroups of cotransformants which had been transformed with either HLA-A1cDNA, or HLA-A2 cDNA, but not both. These results show that the CTLNW38-IVS-1 lysed COS-7 transfectants containing both NY-ESO-1 and HLA-A2exclusively. See FIG. 6. The work also confirmed the specificity of theCTL, since the NY-ESO-1 negative, HLA-A2 positive cells described inExample 9 were positive for other molecules known to be processed topeptides presented by HLA-A2 molecules.

EXAMPLE 12

Once the presenting MHC molecule was identified as HLA-A2, a screeningof the amino acid sequence for NY-ESO-1 was carried out, to identify allpeptides which satisfy this motif, using the model set forth by D'Amaroet al., Human Immunol. 43: 13-18 (1995), and Drijfhout, et al., HumanImmunol. 43: 1-12 (1995) incorporated by reference. Peptidescorresponding to all of the amino acid sequences deduced thereby weresynthesized, using standard techniques, and were then used incytotoxicity assays, following Knuth et al., Proc. Natl. Acad. Sci. USA81: 3511-3515 (1984), incorporated by reference. Specifically, cell lineCEMX721.174.T2 (“T2” hereafter), was used, because it does not processantigens to MHC complexed peptides, thereby making it ideal forexperiments of the type described herein. Samples of T2 cells werelabelled with 100 uCi of Na(⁵¹Cr)O₄, using standard methods, and werethen washed three times, followed by incubation with 10 ug/ml peptideand 2.5 ug/ml of β2-microglobulin. Incubation was for one hour, at roomtemperature. Then responder cells (100 ul of a suspension of CTLNW38-IVS-1) were added, at an effector/target ratio of 90:1, andincubated for four hours in a water saturated atmosphere, with 5% CO₂,at 37° C. Then, plates were centrifuged at 200×g for five minutes, 100ul of supernatant was removed, and radioactivity was measured. Thepercentage of ⁵¹Cr release was determined in accordance with knownstrategies. it was found that the peptides SLLMWITQCFL (SEQ ID NO: 4),SLLMWITQC (SEQ ID NO: 5), and QLSLLMWIT (SEQ ID NO: 6), were the threebest stimulators of CTLs. Comparable results were found when NW-MEL-38and cell lines SK-MEL-37 and MZ-MEL-19 were used as targets, as isshown, supra.

EXAMPLE 13

Studies were carried out to determine if CD4+ helper T cells recognizedcomplexes of MHC-Class II molecules and peptides derived from NY-ESO-1.

Tumor cell line MZ-MEL-19 has been typed as being HLA-DR53 positive.Hence, NY-ESO-1 was screened using Futaki, et al., Immunogenetics42:299-301 (1995), incorporated by reference, which teaches bindingmotifs for HLA-DR53. A total of twenty eight peptides which, in theory,would bind to HLA-DR53, and antigens presenting cells alone.

Peripheral blood lymphocytes (“PBLs”), were isolated from two patientswith metastatic melanoma, who had been typed as HLA-DR53 positive.

The typing was performed using standard, commercially availablereagents. One patient was typed as being positive for HLA-DRB1 (alleles1501-05, 1601-1603, 1605 and 0701), HLA DRB4* (alleles (0101-0103), andDRB5* (alleles 0101), while the second patient was typed as positive forHLA-DRB1* (alleles 1401, 1407, 1408, and 0901), HLA-DRB3* (alleles0201-0203), and DRB4* (alleles 0101-0103). All alleles of HLA-DRB4* arereferred to as HLA-DR53, in accordance with Bodmer, et al., HumanImmunol 34:4-18 (1992), incorporated by reference.

The PBLs were treated with magnetic beads coated with appropriateantibodies to deplete CD4+ and CD8+ T lymphocytes. The remaining cellswere seeded in 24 well plates, at 4×10⁶ cells/well, and were allowed toadhere to the plastic of the wells for 24 hours. Any non-adhering cellswere removed, and the remaining cells were used as antigen presentingcells. These cells were stimulated with GM-CSF (1000 U/ml), and IL-4(1000 U/ml) for 5 days, in 96-well, flat bottom nitrocellulose plates,which had been coated, overnight, at 4° C., with 5 ug/ml of anti-gammainterferon antibodies. Cells were seeded at 3.5×10⁵ cells/well.

The cells were then pulsed with 4 ug/well of test peptide, or 2 ug/wellof the complete NY-ESO-1 protein, as a control.

Then CD4+ T cells were added (1×10⁵) cells/well, in RPMI 1640 mediumaugmented with 10% human serum, L-asparagine (50 mg/l), L-arginine (242mg/l), and L-glutamine (300 mg/l), together with 2.5 ng/ml of IL-2, to afinal volume of 100 ul).

This mixture was incubated for 48 hours at 37° C. in a water saturatedatmosphere. Then, plates were washed, 6 times, with a solution of 0.05%Tween 20/PBS, and then biotinylated anti-interferon gamma antibody, wasadded at 0.5 ug/ml. The antibody was incubated for 2 hours at 37° C.,after which plates were developed with standard reagents, for 1 hour.Substrate 3-ethyl-9-amino carbazole was added, and incubated for 5minutes, with positives being represented by red spots. The number ofred spots/well was indicative of the frequency of CD4+ T lymphocyteswhich recognized complexes of peptide and HLA-DR53, or HLA-DR53 and apeptide processed from recombinant NY-ESO-1. As controls, assays wererun using reagents alone (i.e., CD4+ cells alone, and the stain alone.

The following peptides were found to sensitize the CD4⁺ T lymphocytes torelease gamma interferon.

AADHRQLQLSISSCLQQL (SEQ ID NOS.: 7-9) VLLKEFTVSGNILTIRLTPLPVPGVLLKEFTVSGNI

These three peptides satisfy the motif for binding to HLA-DR53 set forthby Futaki, et al., supra, which is an anchor residue of Tyr, Phe, Trp,or Leu, followed by Ala or Ser three residues downstream.

Additional peptides were found which bind to HLA-DR53.

These peptides are:

GAASGLNGCCRCGARGPE (SEQ ID NOS.: 10-12). SRLLEFYLAMPFATPMEATVSGNILTIRLTAADHRQ.

EXAMPLE 14

A human testicular cDNA expression library was obtained, and screened,with serum from a melanoma patient identified as MZ2. See e.g., parentapplication U.S. Pat. No. 5,804,381 incorporated by reference; also seeU.S. Pat. No. 5,698,396 also incorporated by reference; Sahin, et al.,Proc. Natl. Acad. Sci. USA 92:11810-11813 (1995). This serum had beentreated using the methodology described in these references. Briefly,serum was diluted 1:10, and then preabsorbed with transfected E. colilysate. Following this preabsorption step, the absorbed serum wasdiluted 1:10, for a final dilution of 1:100. Following the finaldilution the samples were incubated overnight at room temperature, withnitrocellulose membranes containing phage plaques prepared using themethodology referred to supra. The nitrocellulose membranes were washed,incubated with alkaline phosphatase conjugated goat anti-human Fc_(γ)secondary antibodies, and the reaction was observed with the substrates5-bromo-4-chloro-3-indolyl phosphate and nitroblue tetrazolium. In asecondary screen, any phagemids which encoded human immunoglobulin wereeliminated.

A total of 3.6×10⁵ pfus were screened, resulting in eight positiveclones. Standard sequencing reactions were carried out, and thesequences were compared to sequence banks of known sequences.

Of the eight clones, two were found to code for known autoimmune diseaseassociated molecules, i.e., Golgin—95 (Fritzler, et al., J. Exp. Med.178:49-62 (1993)), and human upstream binding factor (Chan, et al., J.Exp. Med. 174:1239-1244 (1991)). Three other clones were found to encodefor proteins which are widely expressed in human tissue, i.e., ribosomalreceptor, collagen type VI globular domain, and rapamycin bindingprotein. Of the remaining three sequences, one was found to benon-homologous to any known sequence, but was expressed ubiquitously inhuman tissues (this was found via RT-PCR analysis, but details are notprovided herein). The remaining two were found to be identical to fulllength HOM-MEL-40, described in Ser. No. 08/479,328, while the eighthclone was found to be almost identical to “SSX3,” as described byDeLeeuw, et al., Cytogenet. Cell Genet 73:179-183 (1996), differingtherefrom in only two base pair differences in the coding region. Thesedifferences are probably artifactual in nature; however, the clone alsoincluded a 43 base pair 3′-untranslated region.

EXAMPLE 15

In order to carry out Southern blotting experiments, described infra,the SSX genes were amplified, using RT-PCR.

To do this, two primers were prepared using the published SSX2 sequencei.e., MEL-40A:

5′ - CACACAGGAT CCATGAACGG AGA, (SEQ ID NO: 13), and MEL-40B: 5′ -CACACAAAGC TTTGAGGGGA GTTACTCGTC ATC (SEQ. ID NO: 14)See Crew, et al., EMBO J 14:2333-2340 (1995). Amplification was thencarried out using 0.25 U Taq polymerase in a 25 μl reaction volume,using an annealing temperature of 60° C. A total of 35 cycles werecarried out.

EXAMPLE 16

The RT-PCR methodology described supra was carried out on testiculartotal RNA, and the amplification product was used in southern blottingexperiments.

Genomic DNA was extracted from non-neoplastic tissue samples, and thensubjected to restriction enzyme digestion, using BamHI, Eco RI, orHindIII in separate experiments and then separated on a 0.7% agarosegel, followed by blotting on to nitrocellulose filters. Theamplification products described supra were labeled with ³²P, usingwell-known methods, and the labeled materials were then used as probesunder high stringency conditions (65° C., aqueous buffer), followed byhigh stringency washes, ending with a final wash at 0.2×SSC, 0.2% SDS,65° C.

The Southern blotting revealed more than 10 bands, in each case (i.e.,each of the BamHI, EcoRI, and HindIII digests), strongly suggesting thatthere is a family of SSX genes which contained more than the threeidentified previously. In view of this observation, an approach wasdesigned which combined both PCR cloning, and restriction map analysis,to identify other SSX genes.

EXAMPLE 17

When the sequences of SSX1, 2 and 3 were compared, it was found thatthey shared highly conserved 5′ and 3′ regions, which explained why theoligonucleotides of SEQ ID NOS: 15 and 16 were capable of amplifying allthree sequences under the recited conditions, and suggested that thishomology was shared by the family of SSX genes, whatever its size.Hence, the oligonucleotides of SEQ ID NOS: 3 and 4 would be sufficientto amplify the other members of the SSX gene family.

An analysis of the sequences of SSX1, 2 and 3 revealed that SSX1 and 2contained a BglII site which was not shared by SSX3. Similarly, SSX3contained an EcoRV site not shared by the other genes.

In view of this information, testicular cDNA was amplified, using SEQ IDNOS: 15 and 16, as described supra, and was then subjected to BglIIdigestion. Any BglII resistant sequences were then cloned, sequenced,and compared with the known sequences.

This resulted in the identification of two previously unidentifiedsequences, referred to hereafter as SSX4 and SSX5. A search of theGenBank database found two clones, identified by Accession Number N24445and W00507, both of which consisted of a sequence—tag—derived cDNAsegment. The clone identified by N24445 contained the 3′-untranslatedregion of SSX4, and part of its coding sequence, while the oneidentified as W00507 contained a shorter fragment of the 3′-untranslatedregion of SSX4, and a longer part of the coding sequence. Specifically,N24445 consists of base 344 of SSX4, through the 3-end, plus 319 bases3′ of the stop codon. The W00507 sequence consists of a 99 base pairsequence, showing no homology to SSX genes followed by a regionidentical to nucleotides 280 through the end of SSX4 through 67 bases 3′of the stop codon of the molecule.

Two forms of SSX4 were identified. One of these lacked nucleotides 331to 466 but was otherwise identical to SSX4 as described supra. Further,the shorter form is an alternatively spliced variant.

In Table 1, which follows, the nucleotide and amino acid sequences ofthe 5 known members of the SSX family are compared. One reads the tablehorizontally for nucleotide homology, and vertically for amino acidhomology.

TABLE 1 Nucleotide and amino acid homology among SSX family membersNuclcotide Sequence Homology (%) SSX1 SSX2 SSX3 SSX4 SSX5 SSX1 89.1 89.689.4 88.7 SSX2 78.2 95.1 91.5 92.9 SSX3 77.7 91.0 91.1 92.7 SSX4 79.379.8 80.9 89.8 SSX5 76.6 83.5 84.0 77.7 Amino Acid Sequence Homology (%)Hence, SSX1 and SSX4 share 89.4% homology on the nucleotide level, and79.3% homology on the amino acid level.

When the truncated form of SSX4 is analyzed, it has an amino acidsequence completely different from others, due to alternate splicing andshifting of a downstream open reading frame. The putative protein is 153amino acids long, and the 42 carboxy terminal amino acids show nohomology to the other SSX proteins.

EXAMPLE 18

The genomic organization of the SSX2 genes was then studied. To do this,a genomic human placental library (in lambda phage) was screened, usingthe same protocol and probes described supra in the discussion of thesouthern blotting work. Any positive primary clones were purified, viatwo additional rounds of cloning.

Multiple positive clones were isolated, one of which was partiallysequenced, and identified as the genomic clone of SSX2. A series ofexperiments carrying out standard subcloning and sequencing workfollowed, so as to define the exon—intron boundaries.

The analysis revealed that the SSX2, gene contains six exons, and spansat least 8 kilobases. All defined boundaries were found to observe theconsensus sequence of exon/intron junctions, i.e. GT/AG.

The alternate splice variant of SSX4, discussed supra, was found to lackthe fifth exon in the coding region. This was ascertained by comparingit to the SSX2 genomic clone, and drawing correlations therefrom.

EXAMPLE 19

The expression of individual SSX genes in normal and tumor tissues wasthen examined. This required the construction of specific primers, basedupon the known sequences, and these follow, as SEQ ID NOS: 15-24.

TABLE 2 Gene-specific PCR primer sequences for individual SSX genes SSX1A (5′): 5′-CTAAAGCATCAGAGAAGAGAAGC [nt.44- 66] SSX 1B (3′):5′-AGATCTCTTATTAATCTTCTCAGAAA [nt.440- 65] SSX 2A (5′):5′-GTGCTCAAATACCAGAGAAGATC [nt.41- 63] SSX 2B (3′):5′-TTTTGGGTCCAGATCTCTCGTG [nt.102- 25] SSX 3A (5′):5′-GGAAGAGTGGGAAAAGATGAAAGT [nt.454- 75] SSX 3B (3′):5′-CCCCTTTTGGGTCCAGATATCA [nt.458- 79] SSX 4A (5′):5′-AAATCGTCTATGTGTATATGAAGCT [nt.133- 58] SSX 4B (3′):5′-GGGTCGCTGATCTCTTCATAAAC [nt.526- 48 SSX 5A (5′):5′-GTTCTCAAATACCACAGAAGATG [nt.39- 63] SSX 5B (3′):5′-CTCTGCTGGCTTCTCGGGCCG [nt.335- 54]The specificity of the clones was confirmed by amplifying the previouslyidentified cDNA for SSX1 through SSX5. Taq polymerase was used, at 60°C. for SSX1 and 4, and 65° C. for SSX2, 3 and 5. Each set of primerpairs was found to be specific, except that the SSX2 primers were foundto amplify minute (less than 1/20 of SSX2) amounts of SSX3 plasmid DNA.

Once the specificity was confirmed, the primers were used to analyzetesticular mRNA, using the RT-PCR protocols set forth supra.

The expected PCR products were found in all 5 cases, and amplificationwith the SSX4 pair did result in two amplification products, which isconsistent with alternative splice variants.

The expression of SSX genes in cultured melanocytes was then studied.RT-PCR was carried out, using the protocols set forth supra. No PCRproduct was found. Reamplification resulted in a small amount of SSX4product, including both alternate forms, indicating that SSX4 expressionin cultured melanocytes is inconsistent and is at very low levels whenit occurs.

This analysis was then extended to a panel of twelve melanoma celllines. These results are set forth in the following table.

TABLE 3 SSX expression in melanoma cell lines detected by RT-PCR* SSX1SSX2 SSX3 SSX4 SSX5 MZ2-Mel 2.2 + + − − − MZ2-Mel 3.1 + + − − −SK-MEL-13 − − − − − SK-MEL-19 − − − − − SK-MEL-23 − − − − − SK-MEL-29 −− − − − SK-MEL-30  −*  −* −  −* − SK-MEL-31 − − − − − SK-MEL-33 − − − −− SK-MEL-37 + + − + + SK-MEL-179 − − − − − M24-MET − − − − − *Positive(+) denotes strong expression. Weak positivity was observedinconsistently in SK-MEL-30 for SSX 1, 2, and 4, likely representing lowlevel expression.

EXAMPLE 20

Additional experiments were carried out to analyze expression of themembers of the SSX family in various tumors. To do this, total cellularRNA was extracted from frozen tissue specimens using guanidiumisothiocyanate for denaturation followed by acidic phenol extraction andisopropanol precipitation, as described by Chomczynski, et al, Ann.Biochem 162: 156-159 (1987), incorporated by reference. Samples of totalRNA (4 ug) were primed with oligodT (18) primers, and reversetranscribed, following standard methodologies. The integrity of the cDNAthus obtained was tested via amplifying B-acin transcripts in a 25cycle, standard PCR, as described by Tureci, et al, Canc. Res. 56:4766-4772 (1996).

In order to carry out PCR analyses, the primers listed as SEQ ID NOS:15-24, supra were used, as well as SEQ ID NOS: 25-26, i.e.:

ACAGCATTAC CAAGGACAGC AGCCACC GCCAACAGCA AGATGCATAC CAGGGAC

These two sequences were each used with both SEQ ID NOS: 58 and 18 inorder to detect the SYT/SSX fusion transcript reported for synovialsarcoma by Clark et al, supra, and Crew, et al, supra. The amplificationwas carried out by amplifying 1 μl of first strand cDNA with 10 pMol ofeach dNTP, and 1.67 mN MgCl₂ in a 30 μl reaction. Following 12 minutesat 94° C. to activate the enzyme, 35 cycles of PCR were performed. Eachcycle consisted of 1 minute for annealing (56° C. for SEQ ID NOS: 15 and16; 67° C. for SEQ ID NOS: 17 and 19; 65° C. for SEQ ID NOS: 19 and 20;60° C. for SEQ ID NOS: 21 and 22; 66° C. for SEQ ID NOS: 23 and 24; 60°C. for SEQ ID NOS: 25 and 26 and 26 and 18), followed by 2 minutes at72° C., 1 minute at 94° C., and a final elongation step at 72° C. for 8minutes. A 15 μl aliquot of each reaction was size fractionated on a 2%agarose gel, visualized with ethidium bromide staining, and assessed forexpected size. The expected sizes were 421 base pairs for SEQ ID NOS: 15and 16; 435 base pairs for SEQ ID NOS: 17 and 19; 381 base pairs for SEQID NOS 19 and 20; 413 base pairs for SEQ ID NOS: 21 and 22, and 324 basepairs for SEQ ID NOS: 23 and 24. The conditions chosen were stringent,so as to prevent cross anneling of primers to other members of the SSXfamily. Additional steps were also taken to ensure that the RT-PCRproducts were derived from cDNA, and not contaminating DNA. Eachexperiment was done in triplicate. A total of 325 tumor specimens wereanalyzed. The results are presented in Tables 4 & 5 which follow.

It is to be noted that while most of the SSX positive tumors expressedonly one member of the SSX family, several tumor types showedcoexpression of two or more genes.

Expression of SSX genes in synovial sarcoma was analyzed, because theliterature reports that all synovial sarcoma cases analyzed have beenshown to carry either the SYT/SSX1 or SYT/SSX2 translocation, atbreakpoints flanked by the primer sets discussed herein, i.e., SEQ IDNO: 25/SEQ ID NO: 16; SEQ ID NO: 27/SEQ ID NO: 20; SEQ ID NO. 25/SEQ IDNO: 16; SEQ ID NO: 28/SEQ ID NO: 18. The PCR work described supra showedthat SYT/SSX1 translocations were found in three of the synovial sarcomasamples tested, while SYT/SSX2 was found in one. The one in which it wasfound was also one in which SYT/SSX1 was found. Expression of SSXappeared to be independent of translocation.

TABLE 4 Expression of SSX genes by human neoplasms Tissues at leaseTumor entity tested SSX1 SSX2 SSX3 SSX4 SSX5 one positive % Lymphoma 11— 4 — — — 4 36 Breast cancer 67 5 5 — 10  — 16  23 Endometrial cancer 81 2 — 1 1 1 13 Colorectal cancer 58 3 7 — 9 1 16  27 Ovarian cancer 12 —— — 6 — 6 50 Renal cell cancer 22 — 1 — — — 1 4 Malignant melanoma 3710  13  — 10  2 16  43 Glioma 31 — 2 — 3 — 5 16 Lung cancer 24 1 4 — 1 15 21 Stomach cancer 3 — — — 1 — 1 33 Prostatic cancer 5 — 2 — — — 2 40Bladder cancer 9 2 4 — 2 — 5 55 Head-Neck cancer 14 3 5 — 4 1 8 57Synovial sarcoma 4 — 2 — 1 1 3 75 Leukemia 23 — — — — — 0 0Leiomyosarcoma 6 — — — — — 0 0 Thyroid cancer 4 — — — — — 0 0 Seminoma 2— — — — — 0 0 Total 325 25  50  0 48  7 89 

TABLE 5 Expression pattern of individual SSX genes in SSX-positive tumorsamples.¹ SSX1 SSX2 SSX4 SSX5 Breast Cancer (67 specimens) 51 specimens− − − −  7 specimens − − + −  4 specimens − + − −  2 specimens + − − − 2 specimens + − + −  1 specimen + + + − Melanoma (37 specimens) 21specimens − − − −  5 specimens + + + −  4 specimens − + − −  2 specimens− + + −  1 specimen + − − −  1 specimen + + − −  1 specimen + − + −  1specimen + − + +  1 specimen + + + + Endomet. Cancer (8 specimens)  7specimens − − − −  1 specimen + + + + Glioma (31 specimens) 25 specimens− − − −  3 specimens − + − −  2 specimens − − + − Lung Cancer (24specimens) 19 specimens − − − −  3 specimens − + − −  1 specimen − − − + 1 specimen + + + − Colorectal Cancer (58 specimens) 42 specimens − − −−  7 specimens − + − −  5 specimens − − + −  3 specimens + − + −  1specimen − − + + Bladder Cancer (9 specimens)  4 specimens − − − −  2specimens − + − −  1 specimen − − + −  1 specimen + + − −  1specimen + + + − Head-Neck Cancer (14 specimens)  6 specimens − − − −  2specimens + − − −  2 specimens − + + −  1 specimen − + − −  1 specimen −− + −  1 specimen + + − −  1 specimen − + + + Synovial Sarcoma (4specimens) SSX1 SSX2 SSX4 SSX5 SYT/SSX1 SYT/SSX5 Sy1 − − + − + − Sy2 − +− + + − Sy3 − − − − − + Sy4 − + − − + −

EXAMPLE 21

This example details further experiments designed to identify additionalpeptides which bind to HLA-A2 molecules, and which stimulate CTLproliferation.

First, peripheral blood mononuclear cells (“PBMCs” hereafter) wereisolated from the blood of healthy HLA-A*0201⁺ donors, using standardFicoll-Hypaque methods. These PBMCs were then treated to separateadherent monocytes from non-adherent peripheral blood lymphocytes(“PBLs”), by incubating the cells for 1-2 hours, at 37° C., on plasticsurfaces. Any non-adherent PBLs were cryopreserved until needed infurther experiments. The adherent cells were stimulated to differentiateinto dendritic cells by incubating them in AIMV medium supplemented with1000 U/ml of IL-4, and 1000 U/ml of GM-CSF. The cells were incubated for5 days.

Seven days after incubation began, samples of the dendritic cells(8×10⁵) were loaded with 50 μg/ml of exogenously added peptide. (Detailsof the peptides are provided infra). Loading continued for 2 hours, at37° C., in a medium which contained 1000 U/ml of TNF-α, and 10,000 U/mlIL-1β. The peptide pulsed dendritic cells were then washed, twice, inexcess, peptide free medium. Autologous PBLs, obtained as described,supra, were thawed, and 4×10⁷ PBLs were then combined with 8×10⁵ peptideleaded dendritic cells, (ratio: 50:1), in a medium which contained 5ng/ml of IL-7 and 20 U/ml of IL-2. The cultures were then incubated at37° C.

Lymphocyte cultures were restimulated at 14, 21, and 28 days, in thesame manner as the experiment carried out after 7 days. Cytotoxicityassays were carried out, at 14, 21, and 28 days, using a europiumrelease assay, as described by Blomberg, et al., J. Immunol. Meth. 114:191-195 (1988), incorporated by reference, or the commercially availableELISPOT assay, which measures IFN-γ release.

The peptides which were tested were all derived from the amino acidsequence of NY-ESO-1 as is described in U.S. Pat. No. 5,804,381, toChen, et al., incorporated by reference, or the amino acid sequences ofSSX-4. The peptides tested were:

RLLEFYLAM (SEQ ID NO: 27) and SLAQDAPPL (SEQ ID NO: 28)both of which are derived from NY-ESO-1, and

-   -   STLEKINKT (SEQ ID NO: 29)        derived from SSX-4. The two NY-ESO-1 derived peptides were        tested in ELISPOT assays. The results follow. In summary, three        experiments were carried out. The results are presented in terms        of the number of spots (positives) secured when the HLA-A2        positive cells were pulsed with the peptide minus the number of        spots obtained using non-pulsed cells. As indicated,        measurements were taken at 14, 21 and 28 days.

The following results are for peptide RLLEFYLAM.

Day Measured (Pulsed Cells - Unpulsed Cells) 14 21 28 Expt 1 30 8 * Expt2 22 * 12 Expt 3 6 * 12 *not determined

EXAMPLE 22

In follow up experiments, the T cell cultures described supra weretested on both COS cells which had been transfected with HLA-A*0201encoding cDNA and were pulsed with endogenous peptide, as describedsupra, or COS cells which had been transfected with both HLA-A*0201 andNY-ESO-1 encoding sequences. Again, the ELISPOT assay was used, for bothtypes of COS transfectants. Six different cultures of T cells weretested, in two experiments per culture.

Pulsed with Endogenous Peptide NY-ESO-1 Production Culture 1 Expt 1 6444 Expt 2 44 52 Culture 2 Expt 1 48 45 Expt 2 100 64 Culture 3 Expt 1 2037 Expt 2 16 16 Culture 4 Expt 1 17 40 Expt 2 28 34 Culture 5 Expt 1 3626 Expt 2 4 36 Culture 6 Expt 1 12 62 Expt 2 44 96The fact that the endogenous NY-ESO-1 led to lysis suggests thatNY-ESO-1 is processed to this peptide via HLA-A2 positive cells.

Similar experiments were carried out with the second NY-ESO-1 derivedpeptide, i.e., SLAQDAPPL SEQ ID NO: 28. These results follow:

Pulsed with Endogenous Peptide NY-ESO-1 Production Culture 1 Expt 1 2816 Expt 2 30 14 Culture 2 Expt 1 31 75 Expt 2 30 70 Culture 3 Expt 1 3244

EXAMPLE 23

In further experiments, the specificity of the CTLs generated in theprior experiment was tested by combining these CTLs with COS cells,transfected with HLA-A*0201 encoding sequences, which were then pulsedwith peptide. First, the peptide RLLEFYLAM (SEQ ID NO: 27) was tested,in three experiments, and then SLAQDAPPL (SEQ ID NO: 28) was tested, insix experiments. Europium release was measured, as determined supra, andthe percent of target cells lysed was determined. The results follow:

% LYSIS Peptide Added No Peptide PEPTIDE RLLEFYLAM Expt 1 43 0 Expt 2 80 Expt 3 9 0 PEPTIDE SLAQDAPPL Expt 1 11 0 Expt 2 13 0 Expt 3 13 0 Expt4 21 0 Expt 5 12 0 Expt 6 42 0In additional experiments, the CTLs specific to RLLEFYLAM/HLA-A2complexes also recognized and lysed melanoma cell line SK-Mel-37 whichis known to express both HLA-A2 and NY-ESO-1. This recognition wasinhibited via preincubating the target cells with an HLA-A2 bindingmonoclonal antibody, BB7.2. This confirmed that the CTLs were HLA-A2specificfor the complexes of the peptide and HLA-A2.

EXAMPLE 24

An additional peptide derived from SSX-4, i.e., STLEKINKT (SEQ ID NO:29) was also tested, in the same way the NY-ESO-1 derived peptides weretested. First, ELISPOT assays were carried out, using COS cells whichexpressed HLA-A*0201, and which either expressed full length SSX-4, dueto transfection with cDNA encoding the protein, or which were pulsedwith the peptide. Three cultures were tested, in two experiments. Theresults follow:

Pulsed With Endogenous Peptide NY-ESO-1 Production Culture 1 Expt 1 50100 Expt 2 20 138 Culture 2 Expt 1 8 12 Expt 2 6 14 Culture 3 Expt 1 1547 Expt 2 14 54Further, as with the NY-ESO-1 peptides, specificity of the CTLs wasconfirmed, using the same assay as described supra, i.e., combining theCTLs generated against the complexes with COS cells, transfected withHLA-A*0201, and pulsed with peptide. The europium release assaydescribed supra was used. The results follow:

% LYSIS Peptide Added No Peptide Expt 1 22 0 Expt 2 14 0 Expt 3 46 0Expt 4 16 0As with the NY-ESO-1 derived peptides, CTL recognition was inhibited viapreincubation with the monoclonal antibody BB7.2, confirming specificityof the CTL for complexes HLA-A2 and peptides.

EXAMPLE 25

Additional experiments were carried out on peptides derived from SSX-2i.e, KASEKIFYV (SEQ ID NO: 30), and peptides derived from NY-ESO-1,i.e., SLLMWITQCFL, SLLMWITQC, and QLSLLMWIT (SEQ ID NO: 4-6). In eachcase, the same type of assays as were carried out in examples 8-11 werecarried out. The results were comparable, in that for each peptide, CTLwere generated which were specific for the respective peptide/HLA-A2complex.

EXAMPLE 26

HLA-DR molecules constitute more than 90% of the Class II moleculespresented on the surfaces of antigen presenting cells. Hence, there isinterest in determining peptides which bind to HLA-DR molecules.Further, there is interest in identifying so-called “promiscuous”peptides which bind to subsets of these molecules, as well as peptideswhich are specific for only one particular HLA-DR molecule.

Hammer, et al., J. Exp. Med. 180:2353-2358, the discussion of which isincorporated by reference, presents a methodology for determining suchpeptides. This procedure is also described at www.tepitope.com, and inHammer et al., “Techniques To Identify The Rules Governing Class IIMHC-Peptide Interaction,” in Fernandez et al., ed., “MHC Volume 2 APractical Approach,” Oxford University Press, 1998, pages 197-219,incorporated by reference. Further, in a paper by Sturniolo, et al.,Nature Biotechnology 17: 555-567 (1999), the disclosure of which isincorporated by reference, a method is described for generating peptidesequences which might bind to particular HLA-Class II molecules. Thesemethodologies were used in connection with the amino acid sequence ofNY-ESO-1, set forth supra, and with HLA-DR molecules. These peptideswere then synthesized, using standard methods.

The peptides were then combined with autologous dendritic cells. Thesewere obtained by isolating peripheral blood mononuclear cells (“PBMCs”hereafter), from HLA-DR⁺ donors, using Ficoll-Hypaque methods. ThesePBMCs were then incubated for 1-2 hours at 37° C., on plastic surfaces.Adherent monocytes were then cultured for 5 days in medium that had beensupplemented with IL-4 and GM-CSF. To elaborate, AIMV mediumsupplemented with 1000 U/ml of IL-4, and 1000 U/ml of GM-CSF was used.This incubation stimulates differentiation into dendritic cells.

Samples of dendritic cells (8×10⁵) were then loaded with 50 μg/ml ofendogenously added peptide. The loading proceeded for 2 hours, at 37°C., in medium supplemented with 1000 U/ml of TNF-∝ and 10,000 U/ml ofIL-1β. Peptide pulsed dendritic cells were then washed twice, in excesspeptide free medium. Then, autologous peripheral blood lymphocytes(4×10⁷) were combined with 8×10⁵ peptide loaded dendritic cells (ratioof 50:1), in medium which contained 5 ng/ml of IL-7 and 20 U/ml of IL-2.Incubation was carried out at 37° C.

Cultures were restimulated weekly with peptide loaded, irradiated PBMCs.

The ability of the peptides to form complexes with HLA-DR molecules andto stimulate CD4⁺ cell proliferation was determined by measuring BrdUuptake.

The specificity of the resulting CD4⁺ cells was then tested by combingthem with autologous dendritic cells that had been loaded with peptide,admixed with full length recombinant NY-ESO-1 protein, or with anunrelated protein.

The peptides

Ser Arg Leu Leu Glu Phe Tyr Leu Ala Met Pro Phe Ala Thr (SEQ. ID. NO:31) and Phe Leu Pro Val Phe Leu Ala Gln Pro Pro Ser Gly Gln Arg Arg(SEQ. ID. NO: 32)were used.

The peptides were both found to sensitize and to expand CD4³⁰ cells,from two different healthy donors.

EXAMPLE 27

The same protocol referred to supra was then used to determine peptidesequences from SSX-2 which might bind to HLA-DR molecules. The followingpeptides were identified, and synthesized, using standard methods.

Lys Leu Gly Phe Lys Ala Thr Leu Pro Pro Phe Met Cys Asn Lys; (SEQ. ID.NO: 33) Gln Met Thr Phe Gly Arg Leu Gln Gly Ile Ser Pro Lys Ile Met;(SEQ. ID. NO: 34) Arg Lys Gln Leu Val Ile Tyr Glu Glu Ile Ser Asp ProGlu Glu (SEQ. ID. NO: 35) Lys Ile Phe Tyr Val Tyr Met Lys Arg Lys TyrGlu Ala Met Thr (SEQ. ID. NO: 36) and Phe Gly Arg Leu Gln Gly Ile SerPro Lys Ile Met Pro Lys Lys (SEQ. ID. NO: 37)

These peptides were then tested in competitive binding assays usingpurified HLA-DR molecules.

The assay is described by Falcioni, et ai., Nature Biotechnoiogy 17:562-567 (1999), incorporated by reference. In brief, the assay is a“scintiiiation proximity assay” using HLA-DR molecules that had beenaffinity purified using a monoclonal antibody. The HLA-DR molecules usedwere DR*0101, DR*1501, DR*0301, DR*1101, DR*0701, and DR*0801. Peptideswere tested for their ability to compete with control peptide.

-   -   Tyr Ala Phe Arg Ala Ser Ala Lys Ala        (SEQ. ID. NO: 38) which Falcioni et al., supra show binds to        different HLA-DR molecules. The results are summarized below in        terms of the concentration of test peptide (in nm) needed to        inhibit binding of control peptide by 50%.

HLA-DR Type SEQ. ID. NO: 0101 1501 0301 1101 0701 0801 28 0.32 0.01 30.5 1.6 3 31 55 4 80 0.06 28 0.02 32 36 2.1 100 5 1.2 30

EXAMPLE 28

The results presented supra led to additional experiments using T cellsthat had been isolated from two donors in the manner described supra. Inthese experiments, autologous dendritic cells were prepared, asdescribed, and combined with T cells whose proliferation was determinedin a BrdU assay. In a first set of experiments, the 5 peptides set forthin example 27 were mixed, and the mixture was compared to an equalamount of full length SSX-2 protein, and an irrelevant protein, “TALL.”TALL did not stimulate proliferation at all. The SSX-2 full lengthmolecule provoked just slightly less than 60% proliferation, while thepeptide mixture provoked about 85% proliferation.

Similarly, the two peptides of example 1 were mixed, and compared tofull length NY-ESO-1 protein, the “TALL” molecule, and unloadeddendritic cells. The mixture provoked just under 20% proliferation, andNY-ESO-1 just under 40%. The other two test samples did not provokeproliferation. Also, the fact that CD4+ cells proliferate upon contactto cells that had been pulsed with NY-ESO-1 derived peptides and cellspulsed wtih the full length protein indicates that the peptides areproduced endogenously by cellular processes, i.e., that the full lengthmolecule (NY-ESO-1) is processed to the relevant peptides of SEQ ID NO:26 and 27. The pattern of recognition is specific, in that whendendritic cells were mixed with TALL, there was no recognition, nor wasthere recognition of unpulsed dendritic cells.

The mixture of the two peptides was then compared to each peptide usedalone, and to no peptide. In these experiments, the mixture stimulatedover 70% proliferation while the individual peptides stimulated about40%. No proliferation was observed with no peptide. The T cell donor inthese experiments had not been typed for HLA-DR molecules.

In a second set of experiments, cells taken from a donor who had beentyped as positive for HLA-DR*0101 and HLA-DR*1301 were tested with theindividual peptides of example 27, a mix of the peptides, the fulllength SSX-2 molecule, and the Tall protein described supra. The peptideof SEQ. ID. NO: 33 provoked more proliferation than the other individualpeptides or mixture of these, and performed equally as well as the fulllength molecule.

Six individual experiments were carried out, and in all cases, there wasconsistently greater T cell proliferation induced by dendritic cellsthat had been pulsed with SEQ ID NO: 33 or full length SSX-2 than withany of the other peptides, or the TALL molecule. This indicates that thefull length molecule is processed to at least one Class II molecule, andthat the peptide of SEQ ID NO: 33 can provoke specific CD4⁺ cells.

The foregoing examples show, inter alia, that tumor rejection antigenprecursors, such as NY-ESO-1, SSX-2, and any such molecule identifiedvia the SEREX methodology, is processed to peptides which are presentedby MHC-Class I molecules and MHC-Class II molecules as well. Peptideswhich bind to Class II molecules to form complexes therewith can be usedto stimulate proliferation of CD4+ cells. Further, as was shown herein,molecules which contain such sequences can be used to provoke the CD4+cells as well. The preceding examples show various ways one of ordinaryskill in the art can identify molecules which bind to Class IImolecules. These should not be taken as the only methodologies by whichsuch molecules could be identified. The skilled artisan will be aware ofother approaches to this issue.

A further aspect of the invention is a therapeutic method, wherein oneor more peptides which bind to an MHC-Class II molecule on the surfaceof a patient's tumor cells are administered to the patient, in an amountsufficient for the peptides to bind to the MHC molecules, and provokelysis by T cells. The exemplification given supra for HLA-DR moleculesis by no means the only type of this administration that can be used.Any combination of peptides may be used, such as those for other ClassII molecules. These peptides, which may be used alone or in combination,as well as the entire protein or immunoreactive portions thereof, may beadministered to a subject in need thereof, using any of the standardtypes of administration, such as intravenous, intradermal, subcutaneous,oral, rectal, and transdermal administration. Standard pharmaceuticalcarriers, adjuvants, such as saponins, GM-CSF, and interleukins and soforth may also be used. Further, these peptides and proteins may beformulated into vaccines with the listed material, as may dendriticcells, or other cells which present relevant MHC/peptide complexes.These peptides may also be used to form multimeric complexes ofHLA/peptides, such as those described by Dunbar, et al., Curr. Biol. 8:413-416 (1998), incorporated by reference, wherein fourpeptide/MHC/biotin complexes are attached to a streptavidin or avidinmolecule. Such complexes can be used to identify and/or to stimulate Tcell precursors.

Similarly, the invention contemplates therapies wherein the nucleic acidmolecule which encodes either full length protein, or one or more of therelevant peptides, in polytope form, is incorporated into a vector, suchas an adenovirus based vector, to render it transfectable intoeukaryotic cells, such as human cells.

Assays developed from the results presented supra can also be used inprogression/regression studies. One can monitor the course ofabnormality involving expression of proteins such as NY-ESO-1 or SSX-2,simply by monitoring levels of the protein, its expression, and so forthusing any or all of the methods set forth supra, such as identifyingCD4+ cell presence and/or levels, using antibodies, peptides, etc.

It should be clear that these methodologies may also be used to trackthe efficacy of a therapeutic regime. Essentially, one can take abaseline value for the protein, using any of the assays discussed supra,administer a given therapeutic agent, and then monitor levels of theprotein thereafter, observing changes in protein levels as indicia ofthe efficacy of the regime.

As was indicated supra, the invention involves, inter alia, therecognition of an “integrated” immune response to the molecules ofinterest. One ramification of this is the ability to monitor the courseof cancer therapy. In this method, which is a part of the invention, asubject in need of the therapy receives a vaccination of a typedescribed herein. Such a vaccination results, e.g., in a T cell responseagainst cells presenting MHC/peptide complexes on their cells. Theresponse also includes an antibody response, possibly a result of therelease of antibody provoking proteins via the lysis of cells by the Tcells. Hence, one can monitor the effect of a vaccine, by monitoring animmune response. As is indicated, supra, an increase in antibody titeror T cell count may be taken as an indicia of progress with a vaccine,and vice versa. Hence, a further aspect of the invention is a method formonitoring efficacy of a vaccine, following administration thereof, bydetermining levels of antibodies in the subject which are specific forthe vaccine itself, or a large molecules of which the vaccine is a part.

The effects of a vaccine can also be measured by monitoring the Tcell-response of the subject receiving the vaccine. A number of assayscan be used to measure the precursor frequency of these in vitrostimulated T cells. These include, but are not limited to, chromiumrelease assays, TNF release assays, IFN_(γ) release assays, an ELISPOTassay, and so forth. Changes in precursor T cell frequencies can bemeasured and correlated to the efficacy of the vaccine. Additionalmethods which can be employed include the use of multimeric complexes ofMHC/peptides. An example of such complexes is the tetramericHLA/peptide-biotin-streptavidin system of Dunbar, et al. Curr. Biol. 8:413-416 (1998), incorporated by reference.

The identification of the subject proteins as being implicated inpathological conditions such as cancer also suggests a number oftherapeutic approaches in addition to those discussed supra. Theexperiments set forth supra establish that antibodies are produced inresponse to expression of the protein. Hence, a further embodiment ofthe invention is the treatment of conditions which are characterized byaberrant or abnormal levels of the proteins, via administration ofantibodies, such as humanized antibodies, antibody fragments, and soforth. These may be tagged or labelled with appropriate cystostatic orcytotoxic reagents.

T cells may also be administered. It is to be noted that the T cells maybe elicited in vitro using immune responsive cells such as dendriticcells, lymphocytes, or any other immune responsive cells, and thenreperfused into the subject being treated.

Note that the generation of T cells and/or antibodies can also beaccomplished by administering cells, preferably treated to be renderednon-proliferative, which present relevant T cell or B cell epitopes forresponse, such as the epitopes discussed supra.

The therapeutic approaches may also include antisense therapies, whereinan antisense molecule, preferably from 10 to 100 nucleotides in length,is administered to the subject either “neat” or in a carrier, such as aliposome, to facilitate incorporation into a cell, followed byinhibition of expression of the protein. Such antisense sequences mayalso be incorporated into appropriate vaccines, such as in viral vectors(e.g., Vaccinia), bacterial constructs, such as variants of the knownBCG vaccine, and so forth.

CD4⁺ cells respond to complexes of MHC-Class II molecules and peptides,and MHC-Class II restricted CD4⁺ T cell responses against recombinantNY-ESO-1, presented by autologous cultured dendritic cells have beendetected in melanoma patients. Specifically, CD4⁺ cells were separatedfrom other cells from PBLs or serum samples, using well knowntechniques. Then, they were admixed with dendritic cells which had beenpulsed with NY-ESO-1 protein. Proliferation of CD4⁺ cells was observed,bringing another facet to the integrated immune response discussedherein. Hence, a further aspect of this invention are these CD4⁺ Tcells, peptides which bind to the MHC-Class II molecules, and their usein therapy.

As the examples indicate, ESO-1 is also processed to peptides whichcomplex to MHC Class II molecules, HLA-DR53 in particular.

Other features and applications of the invention will be clear to theskilled artisan, and need not be set forth herein.

The terms and expression which have been employed are used as terms ofdescription and not of limitation, and there is no intention in the useof such terms and expression of excluding any equivalents of thefeatures shown and described or portions thereof, it being recognizedthat various modifications are possible within the scope of theinvention.

1. An isolated nucleic acid molecule consisting of a nucleotide sequencewhich encodes the isolated polypeptide of SEQ ID NO: 32, 33, 34, 35, 36or
 37. 2. The isolated nucleic acid molecule of claim 1 consisting of anucleotide sequence which encodes the isolated polypeptide of SEQ ID NO:32.
 3. The isolated nucleic acid molecule of claim 1 consisting of anucleotide sequence which encodes the isolated polypeptide of SEQ ID NO:33.
 4. The isolated nucleic acid molecule of claim 1 consisting of anucleotide sequence which encodes the isolated polypeptide of SEQ ID NO:34.
 5. The isolated nucleic acid molecule of claim 1 consisting of anucleotide sequence which encodes the isolated polypeptide of SEQ ID NO:35.
 6. The isolated nucleic acid molecule of claim 1 consisting of anucleotide sequence which encodes the isolated polypeptide of SEQ ID NO:36.
 7. The isolated nucleic acid molecule of claim 1 consisting of anucleotide sequence which encodes the isolated polypeptide of SEQ ID NO:37.
 8. A recombinant cell comprising the isolated nucleic acid moleculeof claim
 1. 9. An expression vector comprising the isolated nucleic acidmolecule of claim 1, operably linked to a promoter.
 10. A recombinantcell comprising the expression vector of claim
 8. 11. An isolatednucleic acid molecule consisting of a nucleotide sequence which encodesan isolated polypeptide which binds to at least one MHC Class II, HLA-DRmolecule, selected from the group consisting of HLA-DR*0101,HLA-DR*1501, HLA-DR*0301, HLA-DR*1101, HLA-DR*0701, and HLA-DR*0801,wherein said, isolated polypeptide consists of from 14 to 25 contiguousamino acids found within the tumor rejection antigen precursor NY-ESO-1,which is encoded by the nucleotide sequence set forth in SEQ ID NO: 1.12. A recombinant cell comprising the isolated nucleic acid molecule ofclaim
 11. 13. An expression vector comprising the isolated nucleic acidmolecule of claim 11, operably linked to a promoter.
 14. A recombinant,cell comprising the expression vector of claim
 13. 15. An expression kitcomprising a separate portion of each of (i) an isolated nucleic acidmolecule which encodes the isolated peptide of SEQ ID NO; 32, 33, 34,35, 36 or 37, and (ii) an isolated nucleic acid molecule which encodesan HLA-DR molecule, selected from the group consisting of HLA-DR*0101,HLA-DR*1501, HLA-DR*0301, HLA-DR*1101, HLA-DR*0701, and HLA-DR*0801,wherein said HLA-DR molecule forms a complex with said peptide of (i).